The present invention relates to a method, reagent and test kit for evaluating substances that alter APC response. The invention further relates to a method and a composition for treating a patient with abnormal APC response.
Protein C is the zymogen form of a serine protease known as activated protein C ("APC"), which is a vitamin K-dependent plasma glycoprotein that has anticoagulant and profibrinolytic properties in vivo and in vitro. See, e.g., Esmon, J. Biol. Chem. 264: 4743-46 (1989). Protein C can be activated by the thrombin-thrombomodulin complex on endothelial surfaces.
The anticoagulant effect of APC is the result of proteolytic degradation of both activated factor V ("factor Va"), the cofactor for factor IXa-mediated prothrombin activation, and activated factor VIII ("factor VIIIa"), the cofactor for factor IXa-mediated activation of factor X.
The action of APC can lead to the interruption of thrombin generation and the prevention of activation of the coagulation system. Inhibition of thrombin formation by APC can prevent consequences of (local) thrombin generation such as activation, adhesion and aggregation of platelets; release of vasoactive and proinflammatory material; increase in endothelial permeability; expression of platelet activating factor and granule membrane protein-140 on endothelial cells.
Because the microcirculation is the major site of function of the protein C pathway, the adverse effects of severe protein C deficiency typically first become manifest in the capillaries of the skin, and then progress to the vessels of the eyes, brain and kidneys. Protein C deficiency can result in capillary thrombosis and interstitial bleeding. For example, the protein C deficiency can cause ecchymotic skin lesions which, if untreated, rapidly develop into hemorrhagic bullae with subsequent gangrenous necrosis, sometimes extending to the fascia and leading to autoamputation.
The physiological importance of the protein C pathway is further demonstrated by the occurrence of life-threatening purpura fulminans as a result of excess thrombin formation in homozygous protein C-deficient infants, Dreyfus et al., N. Eng. J. Med. 325: 1565-68 (1991), and by the increased risk of thrombotic events in patients with heterozygous protein C deficiency. Griffin et al., J. Clin. Invest. 68: 1370-73 (1981). Infusion of highly purified APC into animals can prevent arterial or venous thrombus formation in various experimental models, see Gruber et al., Blood, 73: 639-42 (1989), and Schwarz et al., Thromb. Haemostas., 62: 25 (1989), and can prevent E. coli-induced death in a baboon model of sepsis. Taylor et al., J. Clin. Invest. 79: 918-25 (1987).
As described above, one function of APC is to inactivate factor Va. Human blood coagulation factor Va is a heterodimeric glycoprotein that includes a heavy chain (molecular weight of about 105,000 daltons) and a light chain (about 72,000-74,000 daltons). Suzuki et al., J. Biol. Chem. 257: 6556-6564 (1982); Rosing et al., J. Biol. Chem. 268: 21130-21136 (1993). The heavy and light chains are non-covalently associated together by divalent metal ions, such as Ca.sup.2+. The heavy chain region (amino acids 1-709) is composed of two A domains (A1 and A2) associated with a connecting region (amino acids 304-316). The light chain region of the factor (amino acids 1546-2196) includes one A and two C domains (A3, C1, C2). The amino acid sequence of human factor V is described by Jenny et al., Proc. Nat'l Acad. Sci. USA 84: 4846-50 (1987).
The heavy and light chain regions are connected in factor V through the connecting B region, which is removed during activation to form factor Va. Factor Va is a cofactor of activated factor X (factor Xa), that drastically (more than 1000 fold) accelerates the factor Xa catalyzed formation of thrombin from prothrombin.
Proteolytic inactivation of factor Va by APC is one of the key reactions in the regulation (limitation) of thrombin formation. APC-catalyzed cleavage of factor Va is stimulated by the presence of negatively charged membrane surfaces and by protein S. Walker et al., Biochim. Biophys. Acta 571: 333-342 (1979); Suzuki et al., J. Biol. Chem. 258: 1914-1920 (1983); Bakker et al., Eur. J. Biochem. 208: 171-178 (1992); Walker, J. Biol. Chem. 255: 5521-5524 (1980); Solymoss et al., J. Biol. Chem. 263: 14884-14890 (1988). The loss of cofactor activity by factor Va is associated with peptide bond cleavages in its heavy chain at Arg.sup.306, Arg.sup.506, and Arg.sup.679. Kalafatis et al., J. Biol. Chem. 269: 31869-80 (1994), Kalafatis et al., loc. cit. 270: 4054-57 (1995). The physiologic importance of the down-regulation of factor Va activity by APC is underscored by the observation of recurrent thromboembolic events in individuals that are deficient in either protein C or protein S. Griffin et al., J. Clin. Invest. 68: 1370-1373 (1981), Schwarz et al., Blood 64: 1297-1300 (1984), Comp et al., J. Clin. Invest. 74: 2082-2088 (1984).
There have been reports that cleavage at Arg.sup.506 alone in normal factor Va has no effect on cofactor activity. However, this cleavage has previously been thought to be necessary for efficient exposure of cleavage sites at Arg.sup.306 and Arg.sup.679, which are associated with cofactor inactivation. Arg.sup.306 is in the A1 region, Arg.sup.506 is in the A2 region and Arg.sup.679 is in a small part of the B-domain that remains in the heavy chain of factor Va after activation of factor V. In the absence of a membrane surface, APC cleaves factor Va heavy chain to generate a fragment of 75 kD, as well as 28/26 kD and 22/20 kD doublets. Further fragmentation of the heavy chain is possible only in the presence of phospholipids from membrane surfaces. In the presence of such surfaces, APC quickly inactivates factor Va.
There have been recent studies of APC-catalyzed inactivation of factor Va and factor va.sup.R506Q (a mutant where glutamine replaces arginine at position 506) in the presence and absence of phospholipid vesicles, Nicolaes et al., J. Biol. Chem. 270: 21158-66 (1995), or platelet membranes. Camire et al., J. Biol. Chem. 270: 20794-800 (1995). These studies suggest that cleavage of the peptide bond at Arg.sup.306 of factor Va occurs in the absence of phospholipids and that this cleavage is not significantly affected by precleavage of the peptide bond at Arg.sup.506, Accordingly, the cleavage at Arg.sup.306 does not require exposure through binding of factor Va to membranes or cleavage of the molecule at Arg.sup.506.
Suzuki et al., J. Biochem . 96: 455-60 (1984) found that factor Va was inactivated by APC in a purified system depending on the presence of cofactor protein S. Platelet-associated factor Va was incubated with APC and protein S, whereby the rate of factor Va inactivation was about 25 fold higher than without protein S. This stimulating effect of protein S was only minor when thrombin-modified protein S was used as a cofactor for APC. Therefore, this modified protein S was considered to be inefficient. Additionally, an extensive study of the effect of protein S on APC-catalyzed inactivation of platelet factor Va showed only marginal stimulation by protein S. Tans et al., Blood 77: 2641-48 (1991).
In the art, protein S appeared to be a rather poor stimulator of the physiological APC catalyzed factor Va inactivation. Bakker et al., Eur. J. Biochem. 208: 171-78 (1992). The protein S dependent rate enhancement was observed only in reaction mixtures that contained negatively charged phospholipid vesicles. The effect is dependent upon the assay conditions, such as the phospholipid source. It was suggested that in the human system enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. The mechanism of action of protein S in factor Va inactivation is, however, not fully understood.
The anticoagulant effect of APC in vitro is reflected by the fact that APC results in a dose-dependent prolongation of clotting time in assays based on factor Xa or activated partial thromboplastin time (aPTT), provided that a cofactor, the vitamin K-dependent protein S, is present. Walker, J. Biol. Chem. 256: 11128-31 (1981). Amer and Kisiel have observed that the addition of APC to plasma from a patient with thrombosis did not result in the expected prolongation of clotting time. Amer et al., Thrombos. Res., 57: 247-58 (1990). Dahlback demonstrated that the addition of APC to plasma from certain patients with thrombosis but no deficiency of the main inhibitors of clotting (such as antithrombin III, protein C and protein S) did not result in prolongation of the aPTT and suggested a new concept of the pathogenesis of hereditary thrombophilia, referred to as "APC resistance." Dahlback et al., Proc. Nat'l Acad. Sci. USA 90: 1004-08 (1993).
Several laboratories have associated the occurrence of familial thrombophilia in a large group of patients with a poor anticoagulant response to APC (APC resistance or reduced APC response). Dahlback et al. supra. Those patients suffer from thrombotic events despite of having protein S level in a normal range. APC resistance is at least 10 times more common than all other known genetic thrombosis risk factors together and has an allelic frequency of about 2% in the Dutch population. Koster et al., Lancet 342: 1503-1506 (1993). A molecular defect in APC-resistant patients was recently demonstrated to be linked to a single point mutation in the factor V gene that causes an amino acid substitution of Gln for Arg at position 506 ("factor V.sup.R506Q "), which is a position where cleavage occurs during APC-catalyzed inactivation of native factor Va. Bertina et al., Nature 369: 64-67 (1994); Greengard et al., Lancet 343: 1361-62 (1994); Voorberg et al., Lancet 343: 1535-36 (1994); Kalafatis et al., J. Biol. Chem. 269: 31869-80 (1994).
The phrase "APC response" refers to the functional defect of patients who do not respond to the activity of APC in an appropriate way. An extreme case of reduced APC response is APC resistance, which often occurs in patients who are homozygous for a factor V mutation. The APC response therefore may be determined via a functional test such as aPTT in the presence of APC, or a coagulation assay for inactivating factor VIII by APC, which could be designed as a chromogenic assay. See, for example, the determination of sensitivity to APC which is described in published European application No. 65 64 24.
Because of the necessity to treat APC response disorders, there exists a need for methods, reagents and test kits to evaluate the properties of compositions to treat APC response disorders, as well as therapeutic compositions themselves. These needs are satisfied by the present invention.